ACE2-Fc Trap

ABSTRACT

An ACE2-Fc hybrid construct is used as a therapeutic and/or analytic entity to treat an individual infected with a coronavirus or to detect a coronavirus in an analyte. In selected embodiments, the Fc portion of the hybrid construct is an IgA Fc portion, and in still further embodiments the ACE2 portion has a mutation that reduces or abolishes ACE2 catalytic activity.

This application claims priority to our co-pending U.S. 63/016,048,which was filed on 27 Apr. 2020, and which is incorporated by referencein its entirety.

SEQUENCE LISTING

The content of the ASCII text file of the sequence listing namedPAT.005234.US003_ST25, which is 99 KB in size was created on 15 May 2020and electronically submitted via EFS-Web along with the presentapplication. The sequence listing is incorporated by reference in itsentirety.

FIELD

The present disclosure relates to therapeutic and diagnosticcompositions and methods, especially as they relate to hybrid constructsof angiotensin-converting enzyme 2 (ACE2) and an immunoglobulin“fragment crystallizable” (Fc) domain, useful for treatment anddiagnosis of coronaviruses.

BACKGROUND

The background description includes information that may be useful inunderstanding the present disclosure. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

ACE2 is a regulatory carboxypeptidase of the renin-angiotensin hormonesystem and functions as regulator of cardiovascular homeostasis. TheACE2 substrates are angiotensin I (which is converted by ACE2 toangiotensin 1-9) and angiotensin II (which is converted by ACE2 toangiotensin 1-7, a vasodilator). ACE2 also removes the C-terminalresidue from three other vasoactive peptides, neurotensin, kinetensin,and des-Arg bradykinin, but is not active on bradykinin. Additionalsubstrates for ACE2 include apelins, casomorphins, and dynorphin A. Inaddition, ACE2 C-terminus is homologous to collectrin and is responsiblefor regulating expression of the neutral amino acid transporter SL6A19on the cell surface.

Despite its regulatory role in blood pressure, ACE2 is also expressed innumerous other tissues, including lung alveolar epithelial cells,enterocytes of the small intestine, Leydig cells and Sertoli cells, therenal proximal tubule, and testis. ACE2 is a receptor for humancoronaviruses such as SARS-CoV and SARS-CoV-2, as well as humancoronavirus NL63/HCoV-NL63.

Currently, there are no treatments that can reduce coronaviralpathogenic effects. While several antiviral and immunomodulatory drugs(e.g., remdesivir, hydroxychloroquine, azithromycin, immune suppressivesteroids, etc.), have been proposed as COVID-19 treatments, most ofthese will not directly affect the virus or prevent its entry into thehost cells.

A vaccine was prepared comprising a fusion protein with an IgG Fcportion and a viral protein of SARS-CoV as described in US 2010/0150923.However, the therapeutic effectiveness of such a construct was notreported.

In a further example, an ACE2/IgG Fc fusion was made as described inF1000Research 2020, 9:72 and in bioRxiv preprint atdoi.org/10.1101/2020.02.01.929976. However, the extracellular ACE2concentration achieved with such a fusion could have unknown effects onthe body, particularly as the Fc domain prolongs serum half-life.

Thus, even though various compositions and methods of treatment forcoronaviruses are known in the art, they suffer from several drawbacks.Therefore, there remains a need for improved compositions and methodsfor treatment of coronavirus infection.

SUMMARY

Herein are disclosed various compositions of ACE2-IgA Fc fusionconstructs and methods of using and uses of such constructs in thetreatment of viral diseases, especially SARS-CoV-2 disease.Advantageously, these constructs will not only act as decoy receptorsfor coronaviruses by binding to the coronaviral Spike protein, butIgA-Fc domains will also localize them to mucous membranes. In certainembodiments, the ACE2 portion of the ACE2-Fc fusion construct comprisesa mutation that inactivates the protein. Such constructs canadvantageously be used as therapeutics to treat coronavirus.

In one aspect, a soluble recombinant ACE2-Fc hybrid construct comprisesan ACE2 portion coupled to an IgA Fc portion.

In some embodiments, the ACE2 C-terminus is coupled to an IgA FcN-terminus, optionally wherein the ACE2 portion has at least 85%sequence identity to SEQ ID NO:9 and/or wherein the IgA Fc portion hasat least 85% sequence identity to SEQ ID NO:10 or 11. In certainembodiments, the hybrid construct will have at least 85%, for example atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, oreven 100% identity to SEQ ID NO:5. In certain embodiments, the hybridconstruct will have at least 85%, for example at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or even 100% identity toSEQ ID NO:6. In further embodiments, the ACE2 portion has a mutationthat renders the ACE2 portion catalytically inactive. The hybridconstruct may also include a J-chain portion. For example, hybridconstructs may have an amino acid sequence with at least 85% sequenceidentity to SEQ ID NO:7 or 8. In certain embodiments, the hybridconstruct will have at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or even 100% identity to SEQ ID NO:7. In certainembodiments, the hybrid construct will have at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or even 100% identity to SEQ IDNO:8. In still further embodiments, the hybrid construct may furthercomprise an affinity portion or a detectable label coupled to the hybridconstruct. Moreover, the hybrid construct may be formulated in apharmaceutically acceptable carrier (e.g., for inhalation, nasaladministration, or injection).

In certain embodiments, the recombinant nucleic acid that encodes theACE2-Fc hybrid construct as described herein may be an RNA or a DNA.Among other options, the recombinant nucleic acid may be an expressionvector (e.g., having a nucleic acid sequence of SEQ ID NO:3 or SEQ IDNO:4).

In another aspect, methods of treating a coronavirus infection aredisclosed herein. These methods include administering to an individualin need thereof a therapeutically effective amount of an ACE2-Fc hybridconstruct as described herein. Non-limiting examples of coronavirusinfections that can be treated with these methods include those causedby SARS-CoV-2. Most typically, administration comprises nasal orpulmonary administration or intravenous injection.

In yet another aspect, methods of detecting a coronavirus are disclosedherein that include adding a test sample to a test surface to whichACE2-Fc is coupled to thereby bind Spike protein in the sample to theACE2-Fc hybrid, contacting the bound Spike protein with a detectablebinder, and detecting the detectable binder.

For example, the ACE2-Fc hybrid construct may be coupled to the testsurface via a biotin group that is coupled to the ACE2-Fc hybridconstruct, and/or detectable binder is an ACE2-Fc hybrid construct thatis coupled to a detectable label. Among other options, the step ofdetecting the detectable binder may be performed usingelectrochemiluminescence.

Various objects, features, aspects and advantages of the compositionsand methods disclosed herein will become more apparent from thefollowing detailed description of preferred embodiments, along with theaccompanying drawing figures in which like numerals represent likecomponents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are exemplary schematic illustrations of ACE2-IgG₁Fcconstructs.

FIGS. 2A-2B are exemplary schematic illustrations of ACE2-IgAFcJchainconstructs.

FIG. 3 depicts exemplary results for ACE2-IgGiFc small scale productionwith Maxcyte.

FIG. 4 depicts exemplary results demonstrating that ACE2-IgAFcexpression is as efficient as ACE2-IgG1Fc.

FIG. 5 is a schematic representation of an exemplary purificationapproach for ACE2-IgA constructs.

FIG. 6 depicts exemplary results for CaptureSelect IgA columnpurification.

FIG. 7 depicts exemplary results for anion exchange column purification.

FIGS. 8A-8C depict exemplary results demonstrating that Fc avidityimproves binding affinity of ACE2 dimer against 2019-nCoV Spike.

FIG. 9 schematically depicts an exemplary Spike detection assay.

FIG. 10 is a graph showing exemplary results for the assay of FIG. 9.

DETAILED DESCRIPTION

Recited ranges of values herein are merely intended as a shorthandreferring individually to each separate value falling within the range.Unless otherwise indicated herein, each individual value is incorporatedinto the specification as if it were individually recited herein.

As used herein, “administering” a pharmaceutical composition or drugrefers to both direct and indirect administration of the pharmaceuticalcomposition or drug. “Direct administration” is typically performed by ahealth care professional (e.g., physician, nurse, etc.). “Indirectadministration” includes a step of providing or making available thepharmaceutical composition or drug to the health care professional fordirect administration (e.g., via injection, infusion, oral delivery,topical delivery, etc.). “Prognosing” or “predicting” a condition, asusceptibility for development of a disease, or a response to anintended treatment covers the prediction (but not treatment or diagnosisof) the condition, susceptibility and/or response, including the rate ofprogression, improvement, and/or duration of the condition in a subject.

As used in the description herein and throughout the claims that follow,“a,” “an,” and “the” include plural reference unless the context clearlydictates otherwise. Also, “in” includes “in” and “on” unless the contextclearly dictates otherwise. As also used herein, and unless the contextdictates otherwise, “coupled to” includes both direct coupling (in whichtwo elements that are coupled to each other contact each other) andindirect coupling (in which at least one additional element is locatedbetween the two elements). Therefore, “coupled to” and “coupled with”are synonymous.

“Comprises” and “comprising” should be interpreted as referring toelements, components, or steps in a non-exclusive manner, indicatingthat the referenced elements, components, or steps may be present, orutilized, or combined with other elements, components, or steps that arenot expressly referenced. Where the specification or claims refer to atleast one of something selected from the group consisting of A, B, C, .. . and N, the text should be interpreted as requiring only one elementfrom the group, not A plus N, or B plus N, etc.

Various ACE2-Fc hybrid constructs can be prepared and employedtherapeutically and/or diagnostically. In some embodiments, solubleACE2-Fc hybrid constructs as presented herein trap coronavirus when theACE2 portion binds to the coronavirus Spike protein. When used inconjunction with an ACE2-Fc hybrid construct, “soluble” means that theACE2-Fc hybrid construct is not bound to a cell membrane via atransmembrane domain. Soluble ACE2-Fc hybrid constructs can beadministered by injection or inhalation without being bound to a cellmembrane. In further embodiments, the constructs as described herein actas decoy receptors to reduce or even eliminate viral entry.

In other embodiments, the ACE2-Fc hybrid constructs as described hereincan be modified with an affinity portion and/or a detectable portion tofacilitate use in assays to detect or quantify SARS-CoV-2 in abiological fluid (e.g., in a sandwich ELISA).

Most typically, the ACE2-Fc hybrid constructs comprise an ACE2 portioncoupled to an immunoglobulin Fc portion in a single polypeptide chain.As detailed below, the coupling is preferably covalent, such that theACE2 C-terminus joins to an immunoglobulin Fc N-terminus, optionally viaa flexible linker. The ACE2 portion and the immunoglobulin Fc portionmay be of any origin. However, especially preferred ACE2 portions andimmunoglobulin Fc portions will be human portions—i.e., sequences havingat least 85% sequence identity to any one of SEQ ID NOs:9-11. In certainembodiments the ACE2 portion will have at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or even 100% identity to SEQ IDNO:9. Additionally or alternatively, in certain embodiments the Fcportion will have at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or even 100% identity to SEQ ID NO:10, or at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or even100% identity to SEQ ID NO:11.

The ACE2 portion may also be catalytically active. Alternatively, theACE2 portion may carry one or more mutations to reduce or even entirelyabolish ACE2 catalytic activity. It is generally preferred that suchmutants not affect ACE2 binding to the coronaviral Spike protein.Non-limiting examples of coronaviruses include MERS-CoV, SARS-CoV,SARS-CoV-2, and NL63/HCoV-NL63. Suitable ACE2 portions include thosefrom human, mouse, rat, bovine, or yeast (S. cerevisiae) described. Forexample, contemplated ACE2 portions are found under UniProtKB identifierQ9BYF1 (human), Q8R0I0 (mouse), Q5EGZ1 (rat), Q58DD0 (bovine),A0A2J8KU96 (chimpanzee), P21192 (S. cerevisiae), Q56H28 (cat), etc. Mostpreferably, however, the ACE2 sequence will be a human sequence in anyisoform (e.g., isoform 1 or 2).

Additionally or alternatively, the ACE2 portion may be full lengthportion or truncated. Where a truncated portion is used, the ACE2sequence is preferably truncated on the C-terminus. The modified formswill be changed to preserve at least three domains known to interactwith the SARS-CoV Spike glycoprotein. Likewise, ACE2 portions preferablyinclude a leader peptide to allow secretion from a recombinant cellproducing ACE2-Fc constructs. Thus, where a truncation is present on theC terminus, the truncation may remove the sequence motif needed forcleavage by ADAM17 and/or the sequence motif needed for cleavage byTMPRSS11D and/or TMPRSS2.

In further preferred aspects, the ACE2 portion will have one or moreinactivating mutations that reduce or abolish ACE2 proteolytic activitywhile maintaining binding to Spike. Various mutations are known in theart that reduce or abolish ACE2 activity, and all of these are suitable.For example, suitable mutations reduce ACE2 catalytic activity by atleast 20%, or by at least 30%, or by at least 40%, or by at least 50%,or by at least 60%, or by at least 70%, or by at least 80%, or by atleast 90%, or by at least 90%, or even more. In one preferredembodiment, the ACE2 mutation is R273Q, which substantially reducescatalytic activity but retained binding capacity to Spike.

The immunoglobulin Fc portion is an IgG or IgA Fc portion, preferablywith cysteine amino acids in place to dimerize in vivo and in vitro. AnIgA Fc portion may further comprise a J-chain portion coupling it torest of the hybrid construct. An IgG Fc portion enhances serum half-lifeand, in some cases, even binding avidity. On the other hand, an IgA Fcwill preferentially collocate with mucous membrane. As such, the ACE2-Fchybrid construct may be administered via injection/infusion, or inhaled(e.g., as nasal spray or inhaled composition).

All known IgG and IgA sequences are suitable. Particularly preferred IgGand IgA Fc sequences are human, or mammalian (e.g., SEQ ID NOs:10 or11). Most typically the sequences will retain amino acids required forN-glycosylation in a host cell (e.g., CHO cell EC7 cell, etc.).Therefore, suitable Fc portions include at least the second and thirdconstant portions of the heavy chain. Where desired, the IgA Fc portionmay further include a J-chain portion to associate two IgA Fcs. Mosttypically, the J chain will be derived from human or other mammal. Anexemplary J chain sequence can be found at UniProtKB entry P01591.

Additionally, the ACE2 portion and the IgA or IgG Fc portions may bedirectly coupled to each other. Alternatively, they may be coupled via aflexible peptide linker. Typically, such linkers will have between 5 and25 amino acids and all known flexible linkers are deemed appropriate foruse herein. Particularly suitable linkers include a run of glycinesinterspersed with serines (e.g., GGGS or SEQ ID NO:12).

In yet further embodiments, the ACE2-Fc hybrid construct is immobilizedon a carrier. All manners of modifications to permit immobilization aresuitable, e.g., biotinylation, cellulose binding domain, etc. Adetectable label may also be added to the ACE2-Fc hybrid construct toenable in situ detection and/or quantification in a quantitative assay.Suitable labels include luminescent labels, radioisotope labels,enzymatic labels, etc.

ACE2-Fc hybrid constructs can be prepared in numerous manners. Sequencesfor the ACE2 portion and the Fc portions are well known in the art, sothe hybrid constructs can be expressed from recombinant nucleic acids.Most typically, such recombinant nucleic acids may be mRNA fortransfection into producer cells or may be DNA expression vectors(typically mammalian expression vector). Among other suitableconfigurations, expression vectors include those having SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Recombinant cells may comprisethe recombinant nucleic acids or expression vectors described hereinencoding ACE2-Fc hybrid constructs.

Once prepared, therapeutically effective amounts of the ACE2-Fc hybridconstruct may be administered to an individual in need thereof viainhalation or injection, or other suitable route, alone or incombination with other therapeutic agents. Alternatively, modifiedACE2-Fc hybrid constructs may also be used in a diagnostic qualitativeor quantitative assay as described in more detail below.

Examples

Selected exemplary constructs presented herein were expressed in CHO—Susing Maxcyte electroporation. 3.2×10⁸ cells were electroporated inMaxcyte electroporation buffer and cultured in CD Opti CHO media with CDCHO efficient feed for 14 days at 32° C. and 3% CO₂. FIGS. 1A-1B depictexemplary IgG1 containing constructs. FIG. 1A shows an ACE2-IgGiFcconstruct encoded by expression plasmid pWH184Lig93 (SEQ ID NO:1). FIG.1B shows an ACE2_(R273Q)-IgG₁Fc construct (with an ACE2 inactivatingmutation), encoded by expression plasmid pWH185Lig94 (SEQ ID NO:2). FIG.1C depicts a biotinylated version of FIG. 1A for diagnostic tests.Similarly, FIG. 2A shows an ACE2IgAFc construct with a J-chain, encodedby expression plasmid pWH190 2B-2 (SEQ ID NO:3). FIG. 2B shows anACE2_(R273Q)-IgAFc construct with a J-chain encoded by expressionplasmid pWH1913B-1 (SEQ ID NO:4).

ACE2-Fc hybrid constructs were isolated following known isolationprocedures. The ACE2-Fc IgG Hybrid Protein had an amino acid sequence ofSEQ ID NO:5, while the ACE2-Fc IgG R273Q Hybrid Protein (lacking ACE2activity) had an amino acid sequence SEQ ID NO:6. The ACE2-Fc IgA HybridProtein had an amino acid sequence of SEQ ID NO:7, while the ACE2-Fc IgAR273Q Hybrid Protein (lacking ACE2 activity) had an amino acid sequenceSEQ ID NO:8.

Yield and relative purity for a small-scale Maxcyte production of IgG-Fchybrid constructs were in a desirable range (FIG. 3). Table 1 providesnumerical results for the size exclusion chromatography.

TABLE 1 Production Culture % Main on ID Volume (mL) Yield SEC-HPLCACE2-IgG1Fc 30 5.2 90.9 ACE2(R273Q)-IgG1Fc 30 8.4 91.7ACE2-IgG1Fc-Avi-tag 40 6.2 94.5

ACE2-IgAFc expression was as efficient as the expression of ACE2-IgGiFc,with no apparent differences in the mutated form (R273Q) versusnon-mutated form (FIG. 4). Traditional purification processes for IgGare often not effective for IgA, so the hybrid constructs were isolatedusing ion exchange chromatographic media and affinity media selectivefor IgA. FIG. 5 depicts schematically various purification options forthe recombinant polypeptides. FIG. 6 shows exemplary results for theCaptureSelect IgA column purification, while FIG. 7 shows exemplaryresults for anion exchange column purification. FIGS. 6 & 7 show thatthe Fc IgA hybrid constructs could be isolated in a meaningful quantityand at reasonable purity.

The purified ACE2-Fc hybrid constructs were then tested for bindingcapacity and avidity against 2019-n-CoV Spike protein. FIGS. 8A-8Cdepict selected results for binding. SPR demonstrated improved bindingaffinity of ACE2 dimer against 2019-nCoV Spike. Table 2 lists exemplarytest results in numerical format.

TABLE 2 KD Method Ligand Analyte k_(on) (1/Ms) k_(off) (1/s) (nM)Octet-SA sensor Spike-RBD ACE2-IgG1Fc 1.03E+3005 7.89E-05 0.76 Octet-SAsensor Spike-RBD ACE2 (R273Q)-IgG1Fc 1.04E+3005 9.86E-05 0.95 Octet-SAsensor Spike-RBD rACE2 (1-740aa) dimer 5.37E+3004 6.63E-05 1.2 SPR2019-nCoV Spike ACE2 (1-615aa) 1.88E+3005* 2.76E-03* 14.7* monomer

Where modified ACE2-Fc hybrid constructs are used for diagnostic tests,multiple test formats can be chosen. One exemplary system is depicted inFIG. 9 using a biotin tagged ACE2-Fc hybrid construct immobilized to MSDstreptavidin plate and another modified ACE2-Fc hybrid construct fordetection (sulfo-tag MSD label). Results using such test system areshown in FIG. 10. As shown, test systems with ACE2-Fc hybrid constructshave high sensitivity and specificity.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest mannerconsistent with the context.

1. A soluble ACE2-Fc hybrid construct, having an amino acid sequence atleast 85% identical to SEQ ID NO:7 or SEQ ID NO:8.
 2. The soluble hybridconstruct of claim 1, wherein a C-terminus of the ACE2 portion iscoupled to an N-terminus of the immunoglobulin IgA Fc portion, andwherein: a) the ACE2 portion has at least 85% sequence identity to SEQID NO:9; and/or b) the immunoglobulin IgA Fc portion has at least 85%sequence identity to SEQ ID NO:10 or
 11. 3. The soluble hybrid constructof claim 1, wherein the ACE2 portion is catalytically inactive.
 4. Thesoluble hybrid construct of claim 1, further comprising a J-chainportion.
 5. The soluble hybrid construct of claim 1, having an aminoacid sequence at least 95% identity to SEQ ID NO:7 or SEQ ID NO:8. 6.The soluble hybrid construct of claim 1, further comprising a detectablelabel coupled to the hybrid construct.
 7. The soluble hybrid constructof claim 1, formulated in a pharmaceutically acceptable carrier.
 8. Thesoluble hybrid construct of claim 7, wherein the pharmaceuticallyacceptable carrier is formulated for inhalation, nasal administration,or injection.
 9. A recombinant nucleic acid encoding the ACE2-Fc hybridconstruct of claim
 1. 10. The recombinant nucleic acid of claim 9,wherein the nucleic acid is an RNA.
 11. The recombinant nucleic acid ofclaim 9, wherein the nucleic acid is a DNA.
 12. The recombinant nucleicacid of claim 11, wherein the recombinant nucleic acid is an expressionvector.
 13. The recombinant4 nucleic acid of claim 12, having a nucleicacid sequence of SEQ ID NO:3 or SEQ ID NO:4.
 14. A method of treating acoronavirus infection in an individual in need thereof, the methodcomprising: administering to the individual a therapeutically effectiveamount of an ACE2-Fc hybrid construct of claim
 1. 15. The method ofclaim 14, wherein the coronavirus is SARS-CoV-2.
 16. The method of claim14, wherein the administering comprises nasal or pulmonaryadministration or intravenous injection.
 17. A method of detecting acoronavirus, the method comprising: adding a test sample to a surface towhich an ACE2-Fc hybrid construct is coupled, to thereby bindcoronaviral Spike protein to the ACE2-Fc hybrid construct; contactingthe Spike protein that is bound to the ACE2-Fc hybrid construct with adetectable binder; and detecting the detectable binder.
 18. The methodof claim 17, wherein the ACE2-Fc hybrid construct is coupled to the testsurface via a biotin group that is coupled to the ACE2-Fc hybridconstruct.
 19. The method of claim 17 or claim 18, wherein thedetectable binder is an ACE2-Fc hybrid construct that is coupled to adetectable label.
 20. The method of any one of claims 17-19, wherein thedetectable binder includes an electrochemiluminescent moiety.